Actin is a major protein of many eukaryotic cells. One of its functions is to polymerize into filaments which are used as structural elements and tracks for moving cargo around the interior of cells. Actin will polymerize in vitro to form 7 nm filaments just like those seen in cells. FIG. 1a indicates, monomers polymerizing to form a helical filament with concomitant hydrolysis of ATP. The initial phase of polymerization is called nucleation (see FIG. 1b), this phase is characterized by monomers initiating a short filament by coalescence. After nucleation a polymerization phase occurs where the majority of filaments are undergoing net polymerization (see FIG. 1b). When the majority of actin has polymerized a phase called steady state exists, in this phase there is an equilibrium between monomer and polymer such that there is a constant polymer mass (see FIG. 1b).
Actin polymerization is tightly regulated in the cell so that inappropriate polymerization does not interfere with normal cellular processes. Scientists need to be able to measure actin polymerization to determine how the regulation works and to develop drugs to actin interacting proteins. As a system of measuring actin polymerization, pyrene labeled actin was developed in 1983 by Kouyama and Mihashi. Essentially, actin is labeled by reacting pyrene iodoacetamide on actin's number 374 amino acid cysteine. The pyrene actin fluorescences when excited by light at 360 nm wavelength. When the actin polymerizes the pyrene moiety moves into a more hydrophobic pocket and its quantum yield increases, thus pyrene actin polymerization is accompanied by a proportionately higher fluorescence signal (see FIG. 2 for an example). However, prior to this disclosure, pyrene actin has been impossible to store for longer than one month because it's activity would be lost due to protein denaturation. Shelf life was a few days for the unpolymerized monomer or a few weeks for the polymers.
The disclosure described here is a method for stabilizing pyrene labeled actin for extended storage times and an optimal storage buffer thus creating a reproducible source of pyrene actin for various assays. Using the processes described herein, we have stored pyrene actin for greater than 3 years at 4° C. which makes it a viable product for retail.